Rare Case of Paraneoplastic Pemphigus Associated With Prostatic Cancer.

ABSTRACT: The combination of paraneoplastic pemphigus and prostate cancer is extremely unusual and has not been reported yet. Paraneoplastic pemphigus is caused by tumor-induced autoantibodies, which cause damage to the skin and mucosa. The essential treatment is active tumor control. Our patient received a robot-assisted radical prostatectomy and glucocorticoid therapy to improve his condition and relieve his skin lesions.

Surface expression of anti-CD3scfv stimulates locoregional immunotherapy against hepatocellular carcinoma depending on the E1A-engineered human umbilical cord mesenchymal stem cells.

Tumor antigens is at the core of cancer immunotherapy, however, the ideal antigen selection is difficult especially in poorly immunogenic tumors. In this study, we designed a strategy to modify hepatocellular carcinoma (HCC) cells by surface expressing anti-CD3scfv within the tumor site strictly, which depended on the E1A-engineered human umbilical cord mesenchymal stem cells (HUMSC.E1A) delivery system. Subsequently, membrane-bound anti-CD3scfv actived the lymphocytes which lysed HCC cells bypassing the expression of antigens or MHC restriction. First, we constructed the anti-CD3scfv gene driven by human α-fetoprotein (AFP) promoter into an adenoviral vector and the E1A gene into the lentiviral vector. Our results showed that anti-CD3scfv could specifically express on the surface of HCC cells and activate the lymphocytes to kill target cells effectively in vitro. HUMSC infected by AdCD3scfv followed by LentiR.E1A could support the adenoviral replication and packaging in vitro 36 h after LentiR.E1A infection. Using a subcutaneous HepG2 xenograft model, we confirmed that AdCD3scfv and LentiR.E1A co-transfected HUMSC could migrate selectively to the tumor site and produce considerable adenoviruses. The new generated AdCD3scfv infected and modified tumor cells successfully. Mice injected with the MSC.E1A.AdCD3scfv and lymphocytes significantly inhibited the tumor growth compared with control groups. Furthermore, 5-fluorouracil (5-FU) could sensitize adenovirus infection at low MOI resulting in improved lymphocytes cytotoxicity in vitro and in vivo. In summary, this study provides a promising strategy for solid tumor immunotherapy.

[The role of extracellular domain of human 4-1BBL in regulating activities of human peripheral blood lymphocytes in vitro].

AIM: To investigate the role of the extracellular domain of human 4-1BBL (ex4-1BBL) in regulating the in vitro activities of peripheral blood lymphocytes (PBL). METHODS: Viable cells were quantified with Trypan-blue exclusion assay. CytoTox 96 Nonradioactive Cytotoxicity Assay Kit was used for measuring LDH levels of supernatant. ELISA kit was used for measuring IL-2 level. In vitro cytotoxity of PBL combined with anti-CD3/anti-Pgp bispecific diabody plus ex4-1BBL was analyzed with CytoTox 96 nonradioactive method. RESULTS: Ex4-1BBL can increase the proliferation of PBL, reduce cell death, promote IL-2 secretion, and the experimental group with ex4-1BBL showed obviously enhanced cytotoxic effect toward K562/A02 cells. CONCLUSION: Ex4-1BBL may be an important adjuvant for improving activities of PBL.

A role of cell apoptosis in lipopolysaccharide (LPS)-induced nonlethal liver injury in D-galactosamine (D-GalN)-sensitized rats.

Lipopolysaccharide (LPS) is implicated in the pathology of acute liver injury and can induce lethal liver failure when simultaneously administered with D-galactosamine (D-GalN). At the present time, nonlethal liver failure, the liver injury of clinical implication, is incompletely understood following challenge by low-dose LPS/D-GalN. We report here our investigation of the effects of liver injury following a nonlethal dose LPS/D-GalN and the role of apoptosis in this disorder. Blood biochemistry indexes, including those of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL), had risen by 6 h post-LPS/D-GalN injection, reached a peak at 24 h and sustained high levels at 48 h. An abnormal liver appearance was found at 24 and 48 h post-injection. Histopathological changes of hepatic injuries accompanied by hepatocellular death, inflammatory infiltration and hemorrhage began to appear at 6 h and were markedly aggravated at 24 and 48 h. Cell apoptosis was significantly induced by the nonlethal dose LPS/D-GalN challenge, and the apoptotic indexes (AIs) in 24 h- and 48 h-treated rats were approximately 70%, as estimated by the terminal transferase dUTP nick end labeling (TUNEL) assay. The mRNA levels of the inflammatory cytokine IL-1beta rose markedly at 6 h and maintained high levels at 24 and 48 h; however, TNF-alpha levels were normal in the liver tissues of 6-, 24- and 48-h-treated rats. mRNA expression of the damage gene nitric oxide synthase (NOS) was also induced early by the LPS/D-GalN challenge, reaching a peak at 6 h, then gradually decreasing in a stepwise manner; conversely, high expression levels of the apoptosis-inducing gene p53 mRNA were not found in the early post-injection period (6 h) but emerged in the crest-time of liver apoptosis (24 h) and were maintained at this level until the late stage (48 h). We also observed that in 24 h-treated rats, caspase-3, -8, -9 and -12 were markedly activated by LPS/D-GalN challenge. These results suggest that a challenge with low-dose LPS in conjunction with D-GalN can induce nonlethal but marked liver failure, the main morphological feature of which is hepatic apoptosis, which may be associated with a high expression of inducible (i)NOS (early post-injection period) and p53 genes (in the mid and late stages) and at least three apoptosis pathways participate in the pathogenesis.

[Construction of a recombinant Pim3-expressing plasmid and expression and activity thereof: experiment with rats].

OBJECTIVE: To construct a plasmid expressing green fluorescent protein (GFP) and gene of Pim-3, a member of the serine/threonine kinase family, and to investigate the in vivo expression of the construct and its effect on cell apoptosis. METHODS: Pim-3 gene was cloned from myocardium tissues of Wistar rat by RT-PCR and subcloned into GFP-expressing plasmid vector pEGFP-N2 by restriction enzyme. The recombinant plasmid pEGFP-N2/Pim-3 was constructed by T4-ligase and then identified through enzyme digestion and gene sequencing. Thirty-two Wistar rats were randomly divided into 4 equal groups: Group A, as control group; Group B, injected intravenously with Ringer's solution; Group C, injected with blank vector, and Group D, injected with the recombinant plasmid pEGFP-N2/Pim-3. One day later, endotoxin/D-galactoamine (D-GalN) was intraperitoneally injected. 24 hours later the rats were killed. Fluorescence microscopy was used to observe the expression of the reporter gene GFP in the liver tissues. RT-PCR was used to detect the Pim-3 mRNA expression. The hepatic apoptosis was detected by TUNEL assay. The activity of caspase-3 was detected. RESULTS: A 998 bp target cDNA fragment with restriction enzyme sites was amplified and inserted into the multiple clone site of pEGFP-N2 successfully. High expression levels of the target gene Pim-3 and reporter gene GFP were achieved in the rat liver after transfer of the recombinant plasmid. The relative Pim-3 expression level of Group D was 0.49 +/- 0.15, significantly higher than those of Groups A, B, and C (0.06 +/- 0.02, 0, and 0 respectively, all P < 0.01). The apoptotic index of Group D was (4.9 +/- 1.2)%, significantly lower than those of Groups B and C [(72.5 +/- 6.1)% and (69.8 +/- 5.7)% respectively, both P < 0.01]; however, not significantly different from that of Group A [(3.1 +/- 0.7)%]. The activity of caspase-3 of Group D was (76 +/- 27) pmol.min(-1).mg(-1), significantly lower than those of Groups B and C [(147 +/- 55) and (142 +/- 50) pmol.min(-1).mg(-1), respectively, both P < 0.01]; however, not significantly different from that of Group A (60 +/- 15). CONCLUSION: The recombinant plasmid pEGFP-N2/Pim-3 can achieve high expression in living cells and have an inhibitory effect on hepatic apoptosis.

[Cloning and expression of the extracellular domain of 4-1BBL].

RT-PCR was used to clone DNA fragment of the extracellular domain of 4-1BBL from human THP-1 cells (human monocyte), and the expression vector pAYZ4-1BBL was constructed by cloning the extracellular domain of 4-1BBL into the expression vector pAYZ. The extracellular domain of 4-1BBL was expressed in E. coli 16C9 and purified by affinity chromatography. SDS-PAGE and Western blot analysis showed that the relativae molecular weight of soluble 4-1BBL is 22kD which was consistent with the theoretically predicted value. So far as we know, it is the first time that the soluble expression of 4-1BBL in E. coli was achieved 4-1BBL induced a significant release of IL-2 in stimulated Jurkat cells after 48h incubation, especially in the presence of tumor cell. At the same time the apoptosis level of Jurkat cell reduce more than 50%. In conclusion, 4-1BBL may be useful in cancer immunotherapy.

DNA chip analysis of gene expression patterns in poorly-differentiated human stage I lung adenocarcinoma / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To screen genes with abnormal expression in poorly-differentiated human lung adenocarcinoma at stage I with cDNA chip.</p><p><b>METHODS</b>The mRNA was extracted from cancer tissue and matched normal lung tissue, and was labeled by Cy5-dUTP or Cy3-dUTP as probes. Subsequently, the mixed probes were hybridized to the cDNA chip containing 8192 genes. The information was obtained by managing the cDNA chip with ScanArray4000 scanner and GenePix3.0 software.</p><p><b>RESULTS</b>Five hundred and eighty genes were differentially expressed between cancer and normal lung tissue. Compared with normal lung tissue, 405 genes were up-regulated and 175 genes were down-regulated in cancer tissue. These genes are involved in different cell activities such as growth regulation and signal transduction. Among the 66 genes with remarkable differential expression between the two tissues, 39 were up-regulated and 27 down-regulated.</p><p><b>CONCLUSION</b>Many different kinds of genes are possibly involved in the initiation and progression of human lung adenocarcinoma. cDNA chip technique might be a useful method in screening lung cancer implicated genes.</p>